We have established stable mixed donor/host hematopoietic chimerism in dogs by combining sublethal conditioning with 200 cGy total body irradiation (TBI) before and a short course of immunosuppression with mycophenolate mofetil (MMF) and cyclosporine after dog leukocyte antigen (DLA)-identical marrow transplantation. This has allowed transplantation without the severe organ toxicities and myeloablation characteristic of traditional high-dose conditioning regimens. The regimen has been translated successfully into the clinic to treat patients with malignant and nonmalignant diseases. During the last grant period, we showed that the major role of the 200 cGy TBI was to provide host immunosuppression since stable mixed chimerism could also be accomplished in dogs given irradiation limited to the cervical, thoracic, and upper abdominal lymph node chain. Further, we were able to decrease the TBI dose needed for conditioning to 100 cGy in recipient dogs primed with donor peripheral blood mononuclear cells (PBMC) and given the costimulatory blocker cytotoxic T lymphocyte antigen-4 immunoglobulin. Findings suggested that the historic concept of creation of marrow space for allografts by cytotoxic agents was no longer valid and grafts could be established solely with the help of immunosuppressive agents. Subsequent successful transplantation without conditioning in patients with impaired T-cell function lent further support to the new concept of engraftment through shifting of the immunological balance toward donor cells. Here, we seek to validate the new allograft concept and to determine whether specific immunosuppression without or with little toxicity can be substituted for both the broad immunosuppression and the short- and long-term toxicities associated with TBI. Two principal approaches will be taken to accomplish our goals. The first is to reduce host-versus-graft immune responses before marrow grafting by priming recipients with donor PBMC and then eliminating activated donor-reactive T cells by either an antibody against the T-cell activation antigen CD70, the antimetabolite methotrexate, or blockade of two costimulatory pathways, B7:CD28 and CD40:CD154. The second approach is to shift the immunological balance toward the graft using genetically-modified donor T cells which express resistance to MMF.